Thymidylate synthetase is the target of certain clinically useful cancer chemotherapeutic agents; however, little is known concerning its mechanism. The objective of this proposal is the elucidation of mechanistic features of the thymidylate synthetase reaction. Any insight into the mechanism of an enzymic reaction requires a thorough knowledge of the mechanistic features of analogous model (non-enzymic) reactions. Therefore, the first phase of our studies involved the development of congruent chemical models of the enzymic reaction, and a detailed investigation of the mechanisms of the chemical conversion of interest. The second phase involves studies with the purified enzyme in which we shall attempt to verify salient features of our model studies and uncover aspects not observable in the chemical counterparts. We shall examine in detail the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthetase and the accompanying chemical conversion of the cofactor, 5,10-methylenetetrahydrofolate. We have recently demonstrated that this inhibitor forms a covalent bond with the enzyme and acts as a "quasi substrate" which proceeds through a portion of the catalytic sequence before the blockade. We shall also examine the nature of the second binding site we have detected for allosteric effects. The pathway of the thymidylate synthetase catalyzed 5-H exchange of 2'-deoxyuridylic acid shall be investigated, as well as isotope effects on the enzymic reaction. We shall identify the amino acid target of 5-formyl-2'-deoxyuridylate, an affinity labeling agent for the enzyme. We shall complete the syntheses of, and evaluate the enzymic properties of a) 5-deaza-5,10-methylenetetrahydrofolate, b) 8-deazatetrahydrofolate, c) 5-thymidylyltetrahydrofolate, and d) 4-thio-5-fluoro-2'-deoxyuridylate.